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Objective To construct a eukaryotic vector of chicken-derived Wnt3a tagged with EGFP (pCAG-MCs-Wnt3a-EGFP) and investigate the influence to the proliferation and axonal formation of neural precursor cells when Wnt3a was overexpressed during the development of chick embryonic spinal cord. Methods Wnt3a gene was amplified from the total RNA obtained from chick embryonic spinal cord using molecular techniques, then connected with pCAG-MCs-EGFP to construct pCAG-MCs-Wnt3a-EGFP, which was identified by digestion and genetic sequencing. At embryonic day (E) 2.5-3.0, pCAG-MCs-Wnt3a-EGFP (experimental group) and pCAG-MCs-EGFP (control group) were transfected into the chick embryonic spinal cord using in vivo electroporation, respectively. Samples were collected at E4 (5 simples of each groups) and then conducted frozen section. The immunofluorescent staining was performed to detect the expression of Wnt3a and proliferating cell nuclear actigen (PCNA) for analyzing the relationship between Wnt3a and cell proliferation, and observe the axonal formation of neural precursor according to the green fluorescence of Wnt3a protein. Results pCAG-MCs-Wnt3a-EGFP was obtained and its gene sequencing was identical with the Gene bank. Green fluorescence was observed at E4 after pCAG-MCs-Wnt3a-EGFP transformed to chick spinal cord. In transversal section of chick embryonic spinal cord, the results of immunofluorescent staining showed Wnt3a was successfully overexpressed. Meanwhile, the amount of neurons projecting axons was dramatically decreased (n=3, P < 0.01), compared to the control group, concomitant with the significant elevation of PCNA level (n =3, P < 0.01). Conclusion pCAG-MCs-Wnt3a-EGFP is successfully constructed and our study confirmed that Wnt3a plays a vital role in the proliferation and axonal formation of neural precursor cells in the developing chick spinal cord.
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Objective To investigate the radioresistant effects of pprI gene of Deinococcus radiodurans on BALB/c mice.Methods Male BALB/c mice in SPF level were applied for this work.The pEGFP-c1 plasmid and pEGFP-c1-pprI gene recombinant plasmid were transferred into anterolateral muscle of mice with in vivo electroporation technology.The mice were irradiated by 6 Gy 60Co γ-rays in whole body and the mortality of mice was observed within 30 days after irradiation.In addition,the mouse were irradiated with 4 Gy γ-rays and then the peripheral blood cell number,apoptosis rates of thymocyte cells,spleen cells and bone marrow cells were observed in the days of 1,7,14,28 and 35 after irradiation while the histopathological changes of lung and testis were observed in the days 7 and 28 after γ-ray irradiation.Results The highest gene transfection efficiency of muscle cells was obtained in a Plasmid injection amount of 50 μg/50 μl and electric field strength of 200 V/cm.The acute radiation mortality of pEGFP-c1-pprI gene recombinant plasmid transfer group was 30％,lower than that of irradiation group (60.0％) and pEGFP-c1 plasmid transfer group (63.3％) after 6 Gy γ-ray irradiation (x2 =4.90,6.24,P ＜ 0.05).Compared with the irradiation group and pEGFP-c1 plasmid transfer group,the WBC count of pEGFP-c1-pprI gene recombinant plasmid group in peripheral blood of mice was significantly higher in the days of 1,7,14 and 28 (F =16.26,8.10,6.37,10.74,P ＜0.05),PLT count was significantly higher in days of 7 and 14 (F =7.36,5.71,P ＜ 0.05),meanwhile the lymphocyte percentage was increased significantly on the 7th day (F =18.43,P ＜ 0.05) after irradiation.On the other hand,the apoptosis rates of thymocyte cells and bone marrow cells were significantly decreased in the days of 1,7,14,28 and 35 (F =3.88,14.91,14.14,39.86,5.65,P ＜0.05 and F=53.70,11.75,21.78,41.40,4.54,P ＜0.05) while the apoptosis rate of spleen cells was significantly decreased in the days of 1,7,14 and 28 (F =97.95,56.61,33.55,14.71,P ＜0.05) after irradiation.Finally,the radiation histopathological changes of lung and testis of the pEGFP-c1-pprI gene recombinant plasmid group were slight and easy to recover.Conclusions Transfection of pprI gene of Deinococcus radiodurans by in vivo electroporation has significant protective effect on the acute radiation injury in BALB/c mice,which may have important clinical applications.
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Objective To investigate the mechanism of Deinococcus radiodurans pprI gene in enhancing mice radioresistance to γ-rays by transfecting it in vivo.Methods The male Kunming mice were randomly divided into control group,irradiated group,pCMV-HA transfected group and pCMV-HA-pprI transfected group.The pCMV-HA-pprI plasmid contained pprI gene was injected into the muscle of mice which were exposed to total 6 Gy of γ-ray irradiation.After injection,the in vivo gene electroporation technology was used to transfect the pprI gene into the cells,and Western blot was used to identify the PprI protein,mammalian homolog protein Rad51 corresponding to recA gene downstream of pprI,and protein Rad52.Results In the muscle of the mice of transfected pCMV-HA-pprI group,the protein PprI expressed significantly at 1 d post-irradiation,but there was no expression of pprI gene 7 d post-irradiation and in other groups.In the mice of transfected with pCMV-HA-pprI,the expression of Rad51 protein was significantly increased in the lungs at 1,7 and 14 d post-irradiation,and significantly increased in the liver at 1 and 28 d post-irradiation and increased in the kidneys at 1 and 14 d post-irradition.However,there was no obvious change of Rad52 protein expression in the lungs and livers of mice in all groups.Conlusions The prokaryotic gene pprI could act on the mammalian homologisation analogues rad51 gene downstream of recA gene and then increase the expression level of protein Rad51 which results in the enhancement of radioresistance.
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Objective To investigate the effects of in vivo electroporation on plasmid mediated reporter gene expression and immunogenicity of DNA vaccine. Methods Luciferase expression plasmid was administered intramuscularly to BALB/c mice at 8μg and 40μg dosage level through injection with or without eletroporation Luciferase expression level in murine muscle was detected by IVIS imaging system 24 h after injection. DNA vaccine plasmid p1.0-gp1455m carrying codon-optimized env gene of CN54 strain ( HIV-1 CRF07_BC) was administered to mice at dosages of 8μg and 40μg through the two approaches mentioned above. Mice were immunized at week 0,2 and 4. Env-specific immune responses were detected at two weeks post the second and the third vaccinations. Env-specific antibody immune responses were determined by ELISA. Euv-specific cellular immune responses were determined by IFN-γ ELISPOT. Results Luciferase expression level in murine muscle was significantly increased as much as 35 folds through in vivo eletroporation. Results of ELISA and ELISPOT revealed that in vivo eletroporation could significantly enhance both the humoral and cellular immune responses induced by DNA vaccination. The responses induced by electrodelivered p1.0-gp1455m at 8 μg dosage were better than those induced by simple intramuscular injection with 40 μg of plasmid DNA. On the other hand, 2 injections followed by electroporation elicited comparable level of humoral and cellular immune responses with those induced by 3 injections without electroporation. Conclusion In vivo electroporation was capable of enhancing both the plasmid-mediated gene expression and immunogenicity of DNA vaccine.
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In this study, we applied in vivo electroporation(EP) for HBV DNA vaccine administration to improve the cell transfection rate of plasmid DNA and to enhance the immune response. In BALB/c mice (8 mice in each group), the luciferase activity (16170?12533RLU) was 4 digits higher than that of the non EP control (8 02?8 00RLU), the difference between them was very significant ( P